Preparation of 7-dehydrocholesteryl esters



" solvents.

Patented Feb. 2i, 10

PREPARATION or wnnnymto- CHOLESTERYL ESTERS Seymour Bernsteinand Karl'J. Sax, Pearl River,

N. Y., assignor s' to: American Cyanamid' Company, New York, N. Y., a corporation of Maine I No Drawing.

The presentinvention relates to an improved method of preparing 7 -dehydrochole steryl esters.

fl'Ziegler et al. [Ann.', vol. 551-, so (194 2)l'-describes the action of various N,-halogenated mole- He found .3;

cules upon unsaturated substances. that compounds such as N-bromosuccinimide and hljom-trichloroacetanilide can be used to brominate unsaturated compounds such-as'cyclohexene and cholesteryl esters. The bromo compounds, when treated with an amine, split-out the halogen acid as a salt of atheamine and form a compound, having} conjugated double bon'd system. In ace-pending application of one of us, serial Number 714,662, ii1edDecember 6, 1946, a method Iofiobt inin "le hyd o h l s r l es e sw escribed in which the removal of the halogen acids was carried out in the presence of a hydrocarbon solvent. In previous methodsof removing'the halogen acids as anaminesalt, the amine was used as a dehydrohalogenating agent and also as a solvent for the process. The use .of asolvent in this step permits the use of a smalleramount of relatively expensive amines and a larger tamo'unt of the ,relatively cheap hydrocarbon We have found that if a portion of a tertiary amine is added tothe reaction mixture at the ,end of thebromination step while the, mixture is still hot,. increased yields .of v highlynpurified prod- "'uct'ar-iobtained. It maybe that the presence of the amine causes the dehydrohalogenating re- :a'ction begin; to takeplace at once, preventing theformation"ofundesirable cholesteryl derivatives by side reactions. whilelthej applicants do *notwish to be bound by this theory, theyhave found that highly purified products can be obtained in good yield by following this procedure.

. In carrying out the bromination step to obtain 'f-bromocholesteryl esters the most desirable -es- @ters are the benzoate and ,theacetate however,

other esters of cholesterol suchas-the propionate,

.butyrate, nicotinate, chlorobenzoate, nitrobenzolate and vthelike can be used. ,-In carrying out the reaction we prefer-to use a saturated hydrocarbon solvent such as petroleum ether, cyclohexane, and the like or a completely halogenated hydrocarbon such as carbon tetrachloride. Un-

saturated hydrocarbons show a tendency to lower the yield. To insure the absence of unsaturated compounds, we prefer to treat the desired solvent with concentrated sulfuric acid and acidic potassium permanganate, prior to use, to remove unsaturated derivatives. I I

The conversion of the cholesteryl esters to 7- bromocholesteryl esters is .bestcarried out in the Application January 14, 1948, .Serial No. 2, 335

8 Claims; (o1. zen-491.2)

2 presence of: the heat" and light emitted by one or more, electrical: light sources producing predominately -visible lighto These'may'take the form of photofiood or photospot bulbs placed relatively close to the reaction mixture. The reaction is generally completed within the period of from '30 seconds to about 1 hour. At about the time the bromination is complete; a'small amount of a tertiaryamine such as 2,4;6-collidine, dimethylaniline,- pyridine and the reaction mixture; i i

After "allowing-the initial reaction mixture to cool, "thesuccinimide is removed and the solvent is then removed from the reaction mixture by like is added to the hot distillation in vacuo. The residue is dissolved in a second solventsuch" as xyleneytoluene, n-octane, and the like, along with an additional small amount of said tertiary amine. The preferred solvents are those -having a boiling point range between C. and- C. The reaction is completed within a period of from 5 minutesto about 2 hours at the above temperature range.

The process is carried out using various esters hereinbeforementioned. Should '7-dehydroch'o' lesterol itself be desired itcan-be obtainedloy hydro'lyzingthe 7-dehydrocho1esteryl ester with a solution of an 'alkalimetal hydroxide. i i The invention will lie-described with greate particularity in the following. examples which illustrate the preferred method of preparing '7- dehydrocholesteryl esters from the corresponding cholesteryl ester. Parts are by weight unless otherwise indicated.

WW i A Jmixture of 4.9,: g. (0.01 moi) of cholesteryl benzoate,-2.14'g..(0.012 mol) of N-bromosuccini- .mide in.50v ml. petroleum ether (boiling point 55-60 C., purified by shaking successively with concentrated ,sulfuric acid and acidic potassium permanganate to remove unsaturated com;- pounds) was refluxedgo minutes by the heat and light from,two RSP -2 photospot lamps (General Electric Co'.'115volts pla ed two inches from the reactionflas'k. Two ml. of,2,4,6-collidine (boiling point C.) "was added to the still re.- fluxing mixture which was then cooled, filtered by suction to remove 's'uccinimide and any, 'un're} acted N,-bromo'succinimide and distilled in iva'ou'o at -10 to 10C. to remove thev petroleum ether. The residual .oil which has an orange to darlg'red color, m'aycry'stallize somewhat if distillationv carried out-at the lower temperatures for along period of time. To the 7-bromocholesteryl; benzoate-collidinemixture in the reaction flask was added 25 ml. of xylene (boiling point 138-142 0.) previously mixed with 1 ml. of collidine. The solution was refluxed for 15 minutes in a nitrogen atmosphere, during which time part of the collidine crystallized out as the hydrobromide salt. The solution was cooled, anhydrous magnesium sulfate was added and the slurry was filtered by suction. The red solution was distilled in vacuo to remove the xylene. Near the end of the distillation crystals began to appear. The reaction mixture was diluted with 35 ml. of acetone and the mixture cooled quickly to 15 C. The crystals obtained were removed by filtration and washed with acetone. A yield of 2.52 g. of 7-dehydrocholesteryl benzoate having a melting point of 128-132 C., cloudy melt clearing at 158 C. was obtained. An ultra violet spectroscopic analysis showed the product to be at least 80.4% pure. On the basis of this analysis the yield was 41.5%.

The material was recrystallized from acetone several times to achieve maximum purity, wt. 1.36 g., melting point l3914l C., clearing at 188.5 C., 27.9% yield.

Example 2 A mixture of 19.6 g. (0.04 mols) of cholesteryl benzoate, 8.55 g. (0.048 mol) of N-bromosuccinimide and 200 m1. of petroleum ether (boiling point 64-65 C. purified by shaking successively with concentrated sulfuric acid and acidic permanganate to remove unsaturated compounds) was refluxed for 4 minutes by the light and heat of two ESP-2 photospot bulbs placed two inches from the reaction vessel. To the boiling solution was added 8 ml. of 2,4,6-collidine and, after cooling, the mixture was filtered by suction to remove the insoluble succinimide. The solution was then distilled in vacuo at 10 to +10 C. to remove the petroleum ether. A dark red oil containing the collidine and the crude 7-bromocholesterylbenzo ate remained in the flask. The oil was dissolved in a mixture of 100 ml. of xylene (boiling point 138 to 142 C.) and 4 m1. of 2,4,6-co11idine and refluxed in a nitrogen atmosphere for 15 minutes. After cooling, anhydrous magnesium sulfate was added and the slurry was filtered with suction and distilled in vacuo. The partially crystalline red oil was treated with 100 m1. of acetone and cooled in an ice bath. A yield of 12.48 g. of '7-dehydrocholesteryl benzoate having a melting point of 122-128 C., cloudy melt clearing at 152 C. was obtained. spectroscopically the material was 64.5% pure. The yield was 41.3%.

After four recrystallizations from acetone there remained 5.08 -g., melting point 138-140.5 C., clearing at 187.5 0., and an additional 0.84 g., melting point l38-140.5 C., clearing at 185.5 C. was recovered from the mother liquors. The overall yield of pure 7-dehydrocholesteryl benzoate was 30.3%.

Example 3 To 98 g. (0.2 mol) of cholesteryl benzoate, and 42.8 g. (0.24 mol) of N-bromosuccinimide was added 1 liter of carbon tetrachloride and the mixture refluxed for two minutes in the manner described in Example 1. Instead of collidine, 40 ml. of dimethylaniline was used, a small amount being added to the hot reaction mixture before cooling. The reaction mixture of 7-bromocholesteryl benzoate-dimethylaniline and 250 ml. of xylene containing 10 ml. of dimethylaniline was refluxed for 10 minutes. A yield of 32.4 g. of 80.2% pure '7-dehydrocholestery1 benzoate was obtained. This was a yield of 26.8%.

Example 4 teryl benzoate was obtained. This was a yield of Example 5 A reaction mixture identical with that of Examplel was refluxed for 4 minutes and 2 ml. of the tertiary amine, dimethylaniline, added to the hot-mixture. On completing the reaction in the usual manner (Example 1) a yield of 1.84 g., of 82% pure '7-dehydrocholes'teryl benzoate was obtained. This was a yield of 30.9%.

Example 6 v A reaction mixture containing the same quantitles of initial reactants as Example 1, but using a petroleum ether havinga boiling point range of 64-65 Cfwas refluxed for 115 minites. To the hot reaction mixture was added 2 ml. of 2,4,6- collidine and, following the usual procedure (Example 1), a yield of 3.54 g. of 50.4% pure 7-dehydrocholesteryl benzoate was obtained. This was a yield of 36.6%.

Example 7 I The procedure of Example l'wasifollowed except that the refluxing time of the bromination procedure was v10 minutes instead of.80 minutes. Two and eight tenths g. of 7dehydrocholesteryl benzoate was obtained which was 70.2%. jp'ure.

This was a yield of. r

Example'8 When using petroleum ether. having a boiling point of 45 C., refluxing the initial reaction mix.- ture for 20 minutes and continuing theprocedu're as given in Example 1, the yield'of product ob tained'was 3.14 g. of 51.5% purity. This, represented a yield of 33.2%. i

Example-9. A further experiment was carried out using the same quantities of reactants as"infExample but using as a solvent petroleum-ether havingja boiling point' range of vo C. The refluxing time was 4 minutes and the remainingconditions were essentially those of Example 1'. Theyield was 2.81 g. of 81% purity; representing a Wield or A mixture of"4.9 g. (0.01 mol)- cholesj tefry1ben zoate, 2.14 g. (0.012 mol) 'N bromo'suc'cinimide and 50 mlpof' petroleum etherfboilingpoint 6465 C. (purified by shaking successively with mixture was cooled and filteredand thegflltrate was added dropwise over a period of l smin'utes to 35 ml. of refluxing xylene'containingl "ml. of 2,4,6-co1lidinei This gavea flash distillation 'of'the petroleum ether; Then 10 mlfimo're of xylene were added and the'mixture lwas refluxe'dffor 10 minutes, cooled, treated with gamete m gnesium sulfate and filtered through diatom aceous earth. 'Ihe filtrate was evaporated "in" vac'uoand gave a mixture of oil and solid which was worked --up with acetone. The solid was collected by filtra- .-tion and was washed with acetone. A yield of I 3.21 g. of 7-dehydrocholesteryl benzoate was. ob-

tained which on ultra-violet analysis showed a 56.6% pureproduct. This was a yield of 37.3%. The product'produced a cloudy melt at 118 to 125 C. which cleared at 151 C.

Example 11 8.56 g. (0.048 mol) of N-bromosuccinimide and "the mixture was heated to reflux by means of two photospot lamps. The mixture was refluxed for 4 minutes with the lamps as the only source ;,of-;heat ,At the end of thereflux period,'while the mixture was still boiling, 8 m1. of 2,4,6-collidine was added. The mixture was cooled and filtered. The filtrate was evacuated in vacuo in an atmosphere nitrogen with the temperature maintained at'room temperature or slightly below. The residue was treated with about one half of 100 ml. of xylene containing 4 m1. of 2,4,6-cllidine and the distillation was continued for a short time to insure removal of traces of the petroleum ether. The remainder of the collidinexylene mixture was added. The mixture was refluxed for 15 minutes in an atmosphere of nitrogen, cooled and treated with water. The product was worked up in xylene and the extract was washed successively with cold, very dilute hydrochloric acid, water, sodium bicarbonate and water. The extract was dried with anhydrous magnesium sulfate, treated with activated charcoal and filtered through diatomaceous earth. Evapo ration of the brown-yellow xylene solution in a nitrogen atmosphere gave an oil solid which was dissolved in acetone. The solution was put under a nitrogen atmosphere and set in the refri erator overnight. The crystals which separated were collected by filtration and wer washed with a small amount of acetone, previously cooled in an alcoholic-ice bath, and then were washed with a small amount of methanol. A whit solid weighing 5.78 g. was obtained, which was 90.8% pure 7-dehydrocholesteryl acetate. This was a yield of 30.8%. After two recrystallizations from an acetone-methanol mixture, 14.4 g. of substantially pure 'T-dehydrocholesteryl acetate, having a melting point of 128-129.5 C., was obtained. The overall yield of pure product was 24.4%.

We claim:

1. The process of preparing a 7-dehydrocholesteryl ester which comprises heating and illuminating a mixture of a cholesteryl ester and N- bromosuccinimide in a saturated hydrocarbon solvent for a period of from 30 seconds to 1 hour, adding to the hot mixture a tertiary amine, cooling said mixture, filtering, removing said hydrocarbon solvent in vacuo, dissolving the brominated cholesteryl ester in a stable hydrocarbon solvent having a boiling point range between 100 and 150 C. in the presence of a tertiary amine, heating the mixture to within the range of 100 to 150 C. for from 5 minutes to 2 hours, removing said tertiary amine in the form of its acid salt and recovering the '7-dehydrocholesteryl ester.

2. The process of preparing a 7-dehydrocholesteryl ester which comprises heating and illuminating a mixture of a cholesteryl ester and N- bromosuccinimide in a saturated hydrocarbon Solvent for a period of from 30. scondsto 1 hour,

adding to'the hot mixture a tertiary amine, cooling said mixture, filtering, removing said: hydrocarbon solvent in vacuo, dissolving the brominated cholesteryl ester in xylene in the presence of a tertiary amine," heating the mixture to within the range of 100 to 150 C. for from 5 minutes to 2 hours, removing said-.tertiary'amine in the for'mbfitsacid salt and recovering. the 7-dehydrocholesteryl ester.

3. The process of preparing a I-dehydrocholesterylzester which comprises heating and illuminating a mixture of a cholesteryl ester and Nebromosuccinimide in petroleum ether for a period of from .30 seconds to 1 hour, adding to the hotarmixturea tertiary amine, coolingv said mixture, filteringremoving said petroleum ether inuvacuo, dissolving. the brominated cholesteryl ester. in a stable hydrocarbon solvent having a boiling point range between 100 and 150. C. in the presence of a tertiary amine, heating the mixture to within the range of 100 to 150 C. for from 5 minutes to 2 hours, removing said tertiary amine in the form of its acid salt and recovering the 7-dehydrocholesteryl ester.

4. The process of preparing 'Y-dehydrocholesteryl benzoate which comprises heating and illuminating a mixture of cholesteryl benzoate and N-bromosuccinimide in a saturated hydrocarbon solvent for a period of from 30 seconds to 1 hour, adding to the hot mixture a tertiary amine, cooling said mixture, filtering, removing said hydrocarbon solvent in vacuo, dissolving the brominated cholesteryl benzoat in a stable hydrocarbon solvent having a boiling point range between 100 and 150 C. in the presence of a tertiary amine, heating the mixture to within the range of 100 to 150 C. for from 5 minutes to 2 hours, removing said tertiary amine in the form of its acid salt and recovering the 7-dehydrocholesteryl benzoate.

5. The process of preparing 7-dehydrocholesteryl acetate which comprises heating and illuminating a mixture of cholesteryl acetate and N-bromosuccinimide in a saturated hydrocarbon I solvent for a period of from 30 seconds to 1 hour,

adding to the hot mixture a tertiary amine, cooling said mixture, filtering, removing said hydrocarbon solvent in vacuo, dissolving the brominated cholesteryl acetate in a stable hydrocarbon solvent having a boiling point range between and C. in the presence of a tertiary amine, heating the mixture to within the range of 100 to 150 C. for 5 minutes to 2 hours, removing said tertiary amine in the form of its acid salt andrecoverlng the 7-dehydr0cholestery1 acetate.

6. The process of preparing 7-dehydrocholes teryl benzoate which comprises heating and illuminating with an electrical light source, a mixture of cholesteryl benzoate and N-bromosuccinimide in petroleum ether for a period of from 30 seconds to 1 hour, adding to the hot mixture a tertiary amine, cooling said mixture, filtering, removing said petroleum ether in vacuo, dissolving the brominated cholesteryl benzoat in xylene in the presence of a tertiary amine, heating the mixture to within the range of 100 to 150 C. for from 5 minutes to 2 hours, removing said tertiary amine in the form of itsacid salt and recovering the 7-dehydrocholesteryl benzoate.

7. The process of preparing 7-dehydrocholesteryl acetate which comprises heating and illuminating with an electrical light source, a mixture of cholesteryl acetate and N-bromosuccinimide in petroleum ether for a period of from 30 m eegseo from 5 'minutes to 2 hours, removing said vdimethylaniline in the form of its hydrobromide salt and recovering the 7-dehydrocho1esteryl acetate. r a

8 The process of preparing 7-dehydrocholesteryl benzoatewhich comprises heating" andil- Zlnminating with an electrical light source, a-mixture' of cholesteryl benzoate and N-bromosuccinimide in petroleum ether for a period of from 30 seconds to 1 hour, adding to the hot -mixture 2,4,6-collidine, cooling said mixture, filtering,

I removing said petroleum ether in vacuo, dissolving the brominated cholesteryl benzoate in xylene in the presence-of 2,4,6-co'l1idine, heating the mixtu're to within the range of. 100 to .150 far from 5' minutes to 2 hours; removingsaid 2,4,6- collidlne in the form of its'hydrobromide salt and recovering the 7-dehydrocholestery1 benzeate. r

v SEYMOUR BERNSTEIN.

' KARL J. SAX.

REFERENCES CITED The following references are of record inthe' file'of this patient: I l ,v

UNITED STATES PATENTS Number v Name Y at 2,441,091 Vliet May, 4, FOREIGN PATENTS Number Country 7 Date 574,432 Great Britain Jan-. 4',"1 946 

1. THE PROCESS OF PREPARING A 7-DEHYDROCHOLESTERYL ESTER WHICH COMPRISES HEATING AND ILLUMINATING A MIXTURE OF A CHOLESTERYL ESTER AND NBROMOSUCCINIMIDE IN A SATURATED HYDROCARBON SOLVENT FOR A PERIOD OF FROM 30 SECOND TO 1 HOUR, ADDING TO THE HOT MIXTURE A TERTIARY AMINE, COOLING SAID MIXTURE, FILTERING, REMOVING SAID HYDROCARBON SOLVENT IN VACUO, DISSOLVING THE BROMINATED CHOLESTERYL ESTER IN A STABLE HYDROCARBON SOLVENT HAVING A BOILING POINT RANGE BETWEEN 100* AND 150*C. IN THE PRESENCE OF A TERTIARY AMINE, HEATING THE MIXTURE TO WITHIN THE RANGE OF 100* TO 150*C. FOR FROM 5 MINUTES TO 2 HOURS, REMOVING SAID TERTIARY AMINE IN THE FORM OF ITS ACID SALT AND RECOVERING THE 7-DEHYDROCHOLESTERYL ESTER. 